Interferon-induced transmembrane protein 3 (IFITM3) and its antiviral activity.

Infections caused by enveloped viruses require fusion with cellular membranes for viral genome entry. Viral entry occurs following an interaction of viral and cellular membranes allowing the formation of fusion pores, by which the virus accesses the cytoplasm. Here, we focus on interferon-induced transmembrane protein 3 (IFITM3) and its antiviral activity. IFITM3 is predicted to block or stall viral fusion at an intermediate state, causing viral propagation to fail. After introducing IFITM3, we describe the generalized lipid membrane fusion pathway and how it can be stalled, particularly with respect to IFITM3, and current questions regarding IFITM3's topology, with specific emphasis on IFITM3's amphipathic α-helix (AAH) 59V-68M, which is necessary for the antiviral activity. We report new hydrophobicity and hydrophobic moment calculations for this peptide and a variety of active site peptides from known membrane-remodeling proteins. Finally, we discuss the effects of posttranslational modifications and localization, how IFITM3's AAH may block viral fusion, and possible ramifications of membrane composition.

Resin embedded multicycle imaging (REMI): a tool to evaluate protein domains.

Protein complexes associated with cellular processes comprise a significant fraction of all biology, but our understanding of their heterogeneous organization remains inadequate, particularly for physiological densities of multiple protein species. Towards resolving this limitation, we here present a new technique based on resin-embedded multicycle imaging (REMI) of proteins in-situ. By stabilizing protein structure and antigenicity in acrylic resins, affinity labels were repeatedly applied, imaged, removed, and replaced. In principle, an arbitrarily large number of proteins of interest may be imaged on the same specimen with subsequent digital overlay. A series of novel preparative methods were developed to address the problem of imaging multiple protein species in areas of the plasma membrane or volumes of cytoplasm of individual cells. For multiplexed examination of antibody staining we used straightforward computational techniques to align sequential images, and super-resolution microscopy was used to further define membrane protein colocalization. We give one example of a fibroblast membrane with eight multiplexed proteins. A simple statistical analysis of this limited membrane proteomic dataset is sufficient to demonstrate the analytical power contributed by additional imaged proteins when studying membrane protein domains.

A kinetic analysis of calcium-triggered exocytosis.

Although the relationship between exocytosis and calcium is fundamental both to synaptic and nonneuronal secretory function, analysis is problematic because of the temporal and spatial properties of calcium, and the fact that vesicle transport, priming, retrieval, and recycling are coupled. By analyzing the kinetics of sea urchin egg secretory vesicle exocytosis in vitro, the final steps of exocytosis are resolved. These steps are modeled as a three-state system: activated, committed, and fused, where interstate transitions are given by the probabilities that an active fusion complex commits (alpha) and that a committed fusion complex results in fusion, p. The number of committed complexes per vesicle docking site is Poisson distributed with mean n. Experimentally, p and n increase with increasing calcium, whereas alpha and the pn ratio remain constant, reducing the kinetic description to only one calcium-dependent, controlling variable, n. On average, the calcium dependence of the maximum rate (R(max)) and the time to reach R(max) (T(peak)) are described by the calcium dependence of n. Thus, the nonlinear relationship between the free calcium concentration and the rate of exocytosis can be explained solely by the calcium dependence of the distribution of fusion complexes at vesicle docking sites.

A stage-specific preparation to study the Ca(2+)-triggered fusion steps of exocytosis: rationale and perspectives.

Despite groundbreaking work to identify numerous proteins and to focus attention on molecular interactions, the mechanism of calcium-triggered membrane fusion remains unresolved. A major difficulty in such research has been the many overlapping and interacting membrane trafficking steps in the secretory pathway, including those of membrane retrieval. Identifying the specific role(s) of a given protein, beyond its general involvement in exocytosis, has therefore proven problematic. Furthermore, the power of time-resolved optical and electrophysiological assays can be best applied to testing the function of known proteins rather than to the identification of unknown, critical membrane components. The identification of essential membrane constituents requires combined biochemical (molecular) and functional (physiological) analyses. A fully functional, stage-specific physiological membrane preparation would be one direct approach to dissecting the calcium-triggered fusion steps of regulated exocytosis. Herein we review our use of specific minimal membrane preparations consisting of fully primed and docked secretory vesicles, or the isolated vesicles themselves, and characterize the late events of exocytosis, with an aim towards identification of essential molecular components. We have established a functional definition of the fusion complex and its activation by calcium, based on our kinetic analyses. Together with a variety of biochemical and alternate functional assays, we have tested whether the SNARE core complex that is present in our vesicle membranes satisfies the criteria of the functionally defined fusion complex. Rather than a direct fusogenic role, the SNARE complex may promote the calcium sensitivity of fusion, possibly by defining or delimiting a localized, focal membrane fusion site that ensures rapid and efficient exocytosis in vivo.

Sea urchin egg preparations as systems for the study of calcium-triggered exocytosis.

This paper reviews recent work in our laboratory on the mechanism of calcium-triggered exocytosis. Upon echinoderm egg fertilization, cortical secretory vesicle exocytosis is massive and synchronous. By combining physiological and molecular analyses with a variety of purified membrane isolates containing secretory vesicles that fuse to the plasma membrane or each other, we have characterized the final steps of this calcium-triggered exocytosis. Our kinetic analysis led to a functional definition of the fusion complex whose activation by calcium follows Poisson statistics. The properties of this complex are compared with the properties of the heterotrimeric SNARE protein complex that is present in the cortical vesicle system. Our data do not support the hypothesis that this particular heterotrimeric complex is by itself the biological fusogen.

Calcium can disrupt the SNARE protein complex on sea urchin egg secretory vesicles without irreversibly blocking fusion.

The homotypic fusion of sea urchin egg cortical vesicles (CV) is a system in which to correlate the biochemistry and physiology of membrane fusion. Homologues of vesicle-associated membrane protein (VAMP), syntaxin, and SNAP-25 were identified in CV membranes. A VAMP and syntaxin immunoreactive band at a higher apparent molecular mass (approximately 70 kDa) was detected; extraction and analysis confirmed that the band contained VAMP, SNAP-25, and syntaxin. This complex was also identified by immunoprecipitation and by sucrose gradient analysis. VAMP in the complex was insensitive to proteolysis by tetanus toxin. All criteria identify the SNARE complex as that described in other secretory systems. Complexes exist pre-formed on individual CV membranes and form between contacting CV. Most notably, CV SNARE complexes are disrupted in response to [Ca2+]free that trigger maximal fusion. N-Ethylmaleimide, which blocks fusion at or before the Ca2+-triggering step, blocks complex disruption by Ca2+. However, disruption is not blocked by lysophosphatidylcholine, which transiently arrests a late stage of fusion. Since removal of lysophosphatidylcholine from Ca2+-treated CV is known to allow fusion, complex disruption occurs independently from the membrane fusion step. As Ca2+ disrupts rather than stabilizes the complex, the presumably coiled-coil SNARE interactions are not needed at the time of fusion. These findings rule out models of fusion in which SNARE complex formation goes to completion ("zippers-up") after Ca2+ binding removes a "fusion-clamp."

Biochemical and functional studies of cortical vesicle fusion: the SNARE complex and Ca2+ sensitivity.

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca2+ activity curve comparable to exocytosis (CV-PM fusion). Here we show that Sr2+ and Ba2+ also trigger CV-CV fusion, and agents affecting different steps of exocytotic fusion block Ca2+, Sr2+, and Ba2+-triggered CV-CV fusion. The maximal number of active fusion complexes per vesicle, Max, was quantified by NEM inhibition of fusion, showing that CV-CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both Max and the Ca2+ sensitivity of fusion complex activation were comparable to that determined for CV-PM fusion. Using Ca2+-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV-CV and CV-PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca2+, Sr2+, and Ba2+, the complex remains Ca2+- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca2+ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca2+-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca2+ sensitivity of fusion.

The calcium sensitivity of individual secretory vesicles is invariant with the rate of calcium delivery.

Differences in the calcium sensitivity of individual secretory vesicles can explain a defining feature of calcium-regulated exocytosis, a graded response to calcium. The role of the time dependence of calcium delivery in defining the observed differences in the calcium sensitivity of sea urchin egg secretory vesicles in vitro was examined. The calcium sensitivity of individual secretory vesicles (i.e., the distribution of calcium thresholds) is invariant over a range of calcium delivery rates from faster than micromolar per millisecond to slower than micromolar per second. Any specific calcium concentration above threshold triggers subpopulations of vesicles to fuse, and the size of these subpopulations is independent of the time course required to reach that calcium concentration. All evidence supports the hypothesis that the magnitude of the free calcium is the single controlling variable that determines the fraction of vesicles that fuse, and that this fraction is established before the application of calcium. Submaximal responses to calcium cannot be attributed to alterations in the calcium sensitivity of individual secretory vesicles arising from the temporal properties of the calcium delivery. Models that attempt to explain the cessation of fusion using changes in the distribution of calcium thresholds arising from the rate of calcium delivery and/or adaptation are not applicable to this system, and thus cannot be general.

Submaximal responses in calcium-triggered exocytosis are explained by differences in the calcium sensitivity of individual secretory vesicles.

A graded response to calcium is the defining feature of calcium-regulated exocytosis. That is, there exist calcium concentrations that elicit submaximal exocytotic responses in which only a fraction of the available population of secretory vesicles fuse. The role of calcium-dependent inactivation in defining the calcium sensitivity of sea urchin egg secretory vesicle exocytosis in vitro was examined. The cessation of fusion in the continued presence of calcium was not due to calcium-dependent inactivation. Rather, the calcium sensitivity of individual vesicles within a population of exocytotic vesicles is heterogeneous. Any specific calcium concentration above threshold triggered subpopulations of vesicles to fuse and the size of the subpopulations was dependent upon the magnitude of the calcium stimulus. The existence of multiple, stable subpopulations of vesicles is consistent with a fusion process that requires the action of an even greater number of calcium ions than the numbers suggested by models based on the assumption of a homogeneous vesicle population.

Cytosolic alkalinization of vascular endothelial cells produced by an abrupt reduction in fluid shear stress.

Reductions in fluid shear stress produce endothelium-dependent vasoconstriction and promote neointimal hyperplasia, but the intracellular signaling mechanisms involved in these processes are poorly understood. To examine whether decreases in fluid shear stress affect endothelial cytosolic pH, carboxy-seminaphthorhodafluor-1-loaded rat aortic endothelial cells were cultured in glass microcapillary tubes and examined during abrupt reductions in laminar flow. After a 30-minute exposure to a shear stress of 2.7 dyne/cm2 in bicarbonate buffer, the acute reduction of fluid shear stress from 2.7 to 0.3 dyne/cm2 transiently increased cytosolic pH from 7.20+/-0.02 to 7.47+/-0.07 (mean+/-SEM, P<.05 versus control). This was not affected by prior inhibition of the Na+-H+ exchanger with 10 micromol/L ethylisopropylamiloride but was abolished in bicarbonate-free buffer. Recovery from an ammonium chloride prepulse-induced acid load occurred more rapidly when fluid shear stress was abruptly reduced from 2.7 to 0.3 dyne/cm2 after maximal acidification (+0.04+/-0.02 pH unit at 2 minutes) than when shear stress was maintained at 2.7 dyne/cm2 continuously (0.00+/-0.00 pH unit at 2 minutes, P<.05). This accelerated cytosolic pH recovery was dependent on the presence of bicarbonate ion and was blocked by the addition of the exchange inhibitors DIDS (100 micromol/L) and ethylisopropylamiloride or by removal of buffer Na+, indicating that the acute reduction in fluid shear stress activates the extracellular Na+-dependent Cl(-)-HCO3- exchanger and the Na+-H+ exchanger and increases cytosolic pH in vascular endothelial cells.

Poisson-distributed active fusion complexes underlie the control of the rate and extent of exocytosis by calcium.

We have investigated the consequences of having multiple fusion complexes on exocytotic granules, and have identified a new principle for interpreting the calcium dependence of calcium-triggered exocytosis. Strikingly different physiological responses to calcium are expected when active fusion complexes are distributed between granules in a deterministic or probabilistic manner. We have modeled these differences, and compared them with the calcium dependence of sea urchin egg cortical granule exocytosis. From the calcium dependence of cortical granule exocytosis, and from the exposure time and concentration dependence of N-ethylmaleimide inhibition, we determined that cortical granules do have spare active fusion complexes that are randomly distributed as a Poisson process among the population of granules. At high calcium concentrations, docking sites have on average nine active fusion complexes.

Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition.

1. The relation between mitochondrial membrane potential (delta psi m) and cell function was investigated in single adult rat cardiac myocytes during anoxia and reoxygenation. delta psi m was studied by loading myocytes with JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetra-ethylbenzimidazolylcarbocyanine iodide), a fluorescent probe characterized by two emission peaks (539 and 597 nm with excitation at 490 nm) corresponding to monomer and aggregate forms of the dye. 2. De-energizing conditions applied to mitochondria, cell suspensions or single cells decreased the aggregate emission and increased the monomer emission. This latter result cannot be explained by changes of JC-1 concentration in the aqueous mitochondrial matrix phase indicating that hydrophobic interaction of the probe with membranes has to be taken into account to explain JC-1 fluorescence properties in isolated mitochondria or intact cells. 3. A different sensitivity of the two JC-1 forms to delta psi m changes was shown in isolated mitochondria by the effects of ADP and FCCP and the calibration with K+ diffusion potentials. The monomer emission was responsive to values of delta psi m below 140 mV, which hardly modified the aggregate emission. Thus JC-1 represents a unique double sensor which can provide semi-quantitative information in both low and high potential ranges. 4. At the onset of glucose-free anoxia the epifluorescence of individual myocytes studied in the single excitation (490 nm)-double emission (530 and 590 nm) mode showed a gradual decline of the aggregate emission, which reached a plateau while electrically stimulated (0.2 Hz) contraction was still retained. The subsequent failure of contraction was followed by the rise of the emission at 530 nm, corresponding to the monomer form of the dye, concomitantly with the development of rigor contracture. 5. The onset of the rigor was preceded by the increase in intracellular Mg2+ concentration ([Mg2+]i) monitored by mag-indo-1 epifluorescence. Since under these experimental conditions intracellular [Ca2+] and pH are fairly stable, the increase in [Mg2+]i was likely to be produced by a decrease in ATP content. 6. The inhibition of mitochondrial ATPase induced by oligomycin during anoxia was associated with a rapid and simultaneous change of both the components of JC-1 fluorescence, suggesting that delta psi m, instead of producing ATP, is generated by glycolytic ATP during anoxia. 7. The readmission of oxygen induced a rapid decrease of the monomer emission and a slower increase of the aggregate emission. These fluorescence changes were not necessarily associated with the recovery of mechanical function.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of calcium homeostasis in cultured rat aortic endothelial cells by intracellular acidification.

Acidosis produces vasodilation in a process that may involve the vascular endothelium. Because synthesis and release of endothelium-derived vasodilatory substances are linked to an increase in cytosolic calcium concentration ([Ca2+]i), we examined the effect of intracellular acidification on cultured rat aortic endothelial cells loaded either with the pH-sensitive probe carboxy-seminaphthorhodafluor-1 or the Ca(2+)-sensitive fluorescent probe indo 1. The basal cytosolic pH (pHi) of endothelial monolayers in a 5% CO2-HCO3- buffer was 7.27 +/- 0.02 and that in a bicarbonate-free solution was 7.22 +/- 0.03. Acidification was induced either by removal of NH4Cl (delta pHi = -0.10 +/- 0.02), changing from a bicarbonate-free to a 5% CO2-HCO3(-)-buffered solution at constant buffer pH (delta pHi = -0.18 +/- 0.03), or changing from a 5% to a 20% CO2-HCO3- solution (delta pHi = -0.27 +/- 0.07). Regardless of the method used, intracellular acidification increased [Ca2+]i as indexed by indo 1 fluorescence. The increase in [Ca2+]i induced by changing from a 5 to a 20% CO2-HCO3- solution was not significantly altered by removal of buffer Ca2+ either before or after depletion of bradykinin- and thapsigargin-sensitive intracellular Ca2+ stores. Thus intracellular acidification of vascular endothelial cells releases Ca2+ into the cytosol either from pH-sensitive intracellular buffer sites, mitochondria, or from bradykinin- and thapsigargin-insensitive intracellular stores. This Ca2+ mobilization may be linked to endothelial synthesis and release of vasodilatory substances during acidosis.

Different effects of alpha- and beta-adrenergic stimulation on cytosolic pH and myofilament responsiveness to Ca2+ in cardiac myocytes.

alpha-Adrenergic stimulation (alpha-AS) and beta-adrenergic stimulation (beta-AS) of the myocardium are associated respectively with an increase and a decrease in myofilament responsiveness to Ca2+. We hypothesized that changes in cytosolic pH (pH(i)) may modulate these opposite actions of alpha-AS and beta-AS. The effects of alpha-AS (50 microM phenylephrine and 1 microM nadolol) and beta-AS (0.05 microM isoproterenol) on contraction and either cytosolic Ca2+ (Cai) or pH(i) were assessed in adult rat ventricular myocytes bathed in bicarbonate buffer (pH 7.36 +/- 0.05). In cells loaded with the ester derivative (AM form) of indo-1, the 410/490-nm ratio of emitted fluorescence indexed Cai. Myofilament responsiveness to Ca2+ was assessed by the relaxation phase of the length-indo-1 fluorescence relation during a twitch. alpha-AS and beta-AS shifted this relation in opposite directions, indicating that alpha-AS increased and beta-AS decreased myofilament responsiveness to Ca2+. In addition, the positive inotropic action of alpha-AS was associated with an increased Cai transient amplitude in 50% of the myocytes (n = 12), whereas beta-AS always increased Cai (n = 5). In cells loaded with the fluorescent pH(i) probe SNARF-1 AM, the emitted 590/640-nm fluorescence is a measure of pH(i). The effect of alpha-AS on the extent of cell shortening during the twitch (ES) was expressed as the percentage of resting cell length. Both ES and pH(i) were assessed in myocytes bathed in 1.5 mM [Ca2+] and stimulated at 0.5 Hz (control ES, 7.4 +/- 1.5%; control pH(i), 7.11 +/- 0.05; n = 10). alpha-AS enhanced both ES (delta ES, 1.8 +/- 0.6%; p less than 0.05) and pH(i) (delta pH(i), 0.06 +/- 0.01; p less than 0.005), and there was a significant correlation between delta ES and delta pH(i) (r = 0.76, p less than 0.05). A similar effect of alpha-AS on pH(i) was observed in the absence of electrical stimulation (n = 8). The alpha-AS-induced enhancement of ES and pH(i) was abolished by 10 microM ethylisopropylamiloride, a Na(+)-H+ exchange inhibitor (n = 7). In additional experiments, myocytes were preincubated either with 0.2 microM 4 beta-phorbol 12-myristate 13-acetate (n = 8) or with 5 nM staurosporine (n = 8), which have been shown to downregulate and inhibit Ca(2+)-activated phospholipid-dependent protein kinase C, respectively. In either group, alpha-AS had no effect on pH(i) and decreased ES to approximately 60% of control.(ABSTRACT TRUNCATED AT 400 WORDS)

A quasi-elastic light scattering study of smooth muscle myosin in the presence of ATP.

We have investigated the hydrodynamic properties of turkey gizzard smooth muscle myosin in solution using quasi-elastic light scattering (QELS). The effects of ionic strength (0.05-0.5 M KCl) and light chain phosphorylation on the conformational transition of myosin were examined in the presence of ATP at 20 degrees C. Cumulant analysis and light scattering models were used to describe the myosin system in solution. A nonlinear least squares fitting procedure was used to determine the model that best fits the data. The conformational transition of the myosin monomer from a folded form to an extended form was clearly demonstrated in a salt concentration range of 0.15-0.3 M KCl. Light chain phosphorylation regulates the transition and promotes unfolding of the myosin. These results agree with the findings obtained using sedimentation velocity and electron microscopy (Onishi and Wakabayashi, 1982; Trybus et al., 1982; Trybus and Lowey, 1984). In addition, we present evidence for polymeric myosin coexisting with the two monomeric myosin species over a salt concentration range from 0.05 to 0.5 M KCl. The size of the polymeric myosin varied with salt concentration. This observation supports the hypothesis that, in solution, a dynamic equilibrium exists between the two conformations of myosin monomer and filaments.

Cytosolic pH measurements in single cardiac myocytes using carboxy-seminaphthorhodafluor-1.

This study examines the use of carboxy-seminaphthorhodafluor-1 (C-SNARF-1) as an indicator of cytosolic pH in isolated rat cardiac myocytes. The emission spectrum of C-SNARF-1 when excited at 530 nm contains two well-separated peaks at approximately 590 and 640 nm, corresponding to the acidic and basic forms of the indicator. This spectral feature allows the indicator to be used in the single excitation, dual emission ratio mode. When C-SNARF-1 is loaded into rat cardiac myocytes as the membrane permeant ester derivative, C-SNARF-1/AM, the indicator localizes within the cytosol with virtually no partitioning into the mitochondria. C-SNARF-1 does not load into isolated mitochondria in suspension. There was no evidence for the presence of non-deesterified C-SNARF-1 within the cells. C-SNARF-1 can be calibrated in situ using a technique that abolishes all transsarcolemmal pH gradients. A 0.7-unit shift in the apparent pK (pKapp = pK-log10) between the in vitro calibration and the in situ calibration is consistent with a change in beta (I640 to pH 9/I640 at pH 5) in the cytosolic environment (beta in situ/beta in vitro = 0.21) and not a change in the true pK of the indicator. The contribution of cellular autofluorescence to the total signal can be made negligible. There is no effect of C-SNARF-1 on the contractile properties of rat cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicardipine prevents calcium loading and "oxygen paradox" in anoxic single rat myocytes by a mechanism independent of calcium channel blockade.

The protective effect of nicardipine (1 and 4 microM) against reoxygenation injury was studied in an unstimulated rat single myocyte oxygen paradox model in comparison with control (no drug) or nifedipine (1 microM). Either concentration of nicardipine was strongly protective, approximately doubling the duration of ATP depletion (rigor) that cells could withstand without undergoing hypercontracture when reoxygenated. Nifedipine (1 microM), which matched the negative inotropic effect of nicardipine (4 microM) (as measured by extent of shortening when stimulated), had no protective effect against reoxygenation injury. Neither drug affected the time to rigor, which is a measure of the rate at which the resting cell consumes its endogenous glycogen stores during anaerobic metabolism. Intracellular calcium, measured with the fluorescent probe indo-1, which partitions into both cytosol and mitochondria, rose progressively throughout the rigor period. This rise in calcium was almost totally suppressed by nicardipine (1 microM) but was unaffected by nifedipine. We conclude that nicardipine possesses a direct protective effect on the myocardium not shared by all dihydropyridines. This effect is associated with the prevention of intracellular, and probably mitochondrial, calcium loading but is probably not due to blockade of the L-type calcium channel or reduction of metabolic rate.

A cellular mechanism for impaired posthypoxic relaxation in isolated cardiac myocytes. Altered myofilament relaxation kinetics at reoxygenation.

Single, isolated rat ventricular myocytes were made hypoxic for 10 minutes and then reoxygenated. During hypoxia, there was a marked abbreviation of the mechanical twitch, without a decrease in its amplitude. Immediately after reoxygenation, both the time to peak shortening and the duration of relaxation were markedly prolonged, and they remained prolonged for 10-50 minutes. The alterations in contraction and relaxation were not associated with any change in the time course of either the transmembrane action potential or the cytosolic calcium transient, as recorded with the fluorescent probe indo 1. Intracellular pH, measured with a fluorescent probe (carboxyseminaphthorhodofluor), showed an acid shift during hypoxia and an alkaline rebound immediately after reoxygenation. The time courses of intracellular pH and contraction duration were not parallel during hypoxia or reoxygenation, and simulation of the alkaline pH shift by lowering PCO2 or superfusing NH4Cl (in the absence of exposure to hypoxia) did not quantitatively reproduce the prolongation of relaxation seen after reoxygenation. The prolongation of contraction after reoxygenation could be overridden by the beta-adrenergic agonist isoproterenol or the nonenzymatic phosphatase butanedione monoxime. We conclude that delayed relaxation after reoxygenation exists at the single cell level and is due to an alteration of the properties of the myofilaments. Intracellular pH is not the primary mediator of this alteration. We speculate that alteration of intracellular inorganic phosphate or covalent modification of the myofilaments might be involved.

Laser backscatter studies of intracellular Ca2+ oscillations in isolated hearts.

We measured intensity fluctuations of 633 nm laser light backscattered from the epicardial surface of isolated, perfused rat and rabbit hearts. Scattered light intensity fluctuations (SLIF) were detected from verapamil-arrested rat hearts. The frequency of SLIF was increased by maneuvers that raise intracellular calcium. SLIF were abolished by removal of extracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and by blockade of sarcoplasmic reticulum calcium release by ryanodine. SLIF were not accompanied by any surface electro-cardiogram and were not abolished by 144 mM extracellular potassium. SLIF were absent in rabbit hearts under base-line conditions but could be provoked by calcium loading using zero potassium and ouabain. We conclude that backscatter SLIF monitor the microscopic motion caused by intracellular calcium oscillations in the intact heart. We measured SLIF from rat hearts during 60 min of global ischemia at 30 degrees C, followed by reflow. Ischemia reduced SLIF frequency to zero within 30 min. Reflow caused an overshoot of SLIF frequency to as much as five times control, suggesting that reflow causes major calcium overload of cells that are at least transiently viable.

Form birefringence of muscle.

We investigate the sensitivity of measurements of muscle birefringence to cross-bridge dynamics in the resting, active, and rigor states. The theory of form birefringence is reviewed, and an optical model is constructed for the form birefringence of muscle. Values for the parameters in the model are selected or deduced from the literature. As an illustration of the use of the model, plausible distributions for the orientations of cross-bridges in the resting, active, and rigor states are constructed using a model for cross-bridge dynamics suggested by Huxley and Kress (1985). The general magnitude of the predictions of our model is comparable with that of published measurements of muscle birefringence. However, the precise values of the predicted birefringence for the resting, active, and rigor states are sensitive to the assumed orientations of cross-bridges. We also investigate the dependence of muscle birefringence on sarcomere length and on disorder in the orientation of the myofilament array. We conclude that measurements of muscle birefringence can play a useful role in distinguishing between proposed models of cross-bridge dynamics.